Method for purifying and concentrating bovine parainfluenza virus type 3

Abstract

The invention relates to the technical field of virus purification and concentration, in particular to a purification and concentration method of bovine parainfluenza virus type 3. The purification and concentration method of the bovine parainfluenza virus type 3 comprises the following steps: purifying and concentrating virus liquid by adopting a hollow fiber column; the purification and concentration comprises three times of washing and filtering, the volumes of washing and filtering liquid used for the three times of washing and filtering are sequentially decreased, the volume of the washing and filtering liquid used for the first time of washing and filtering is 5-10 times of the concentration volume, the volume of the washing and filtering liquid used for the second time of washing and filtering is 2-5 times of the concentration volume, and the volume of the washing and filtering liquid used for the third time of washing and filtering is 1-2 times of the concentration volume. The method aims at the bovine parainfluenza virus type 3 virus liquid subjected to suspension culture, can effectively remove impure protein, has a high virus recovery rate, can significantly improve the virus titer of the virus liquid, greatly reduces the impure protein content of the prepared concentrated virus liquid, and can effectively save the washing and filtering time and reduce the cost compared with three times of large-volume washing and filtering. Good application prospects are realized.

Description

Technical Field

[0001] The invention relates to the technical field of virus purification and concentration, and in particular to a method for purifying and concentrating bovine parainfluenza virus type 3. Background Art

[0002] Bovine parainfluenza virus type 3 (BPIV3) is a single-stranded, negative-sense, enveloped RNA virus and one of the main pathogens that cause acute respiratory diseases in cattle and other ruminants. Vaccination is an important means of preventing bovine parainfluenza virus type 3, and the purification and concentration of virus fluid is a key step in the vaccine production process.

[0003] At present, the suspension culture process of viruses has become the mainstream production method for biological products such as vaccines and antibodies. The suspension culture process has higher controllability in the antigen production process. The antigens produced not only have considerable output, but also have small differences between antigen batches, which steadily improves product quality. However, the content of impure proteins in antigens produced by the suspension culture process is very high, which is dozens to hundreds of times the content of impure proteins in antigens produced by adherent culture. The presence of impure proteins seriously affects the safety of vaccines. Therefore, it is of great significance to develop an efficient purification and concentration method for suspension cultured bovine parainfluenza virus type 3 virus liquid. Summary of the invention

[0004] The invention provides a method for purifying and concentrating bovine parainfluenza virus type 3.

[0005] The present invention develops an efficient purification and concentration method for bovine parainfluenza virus type 3 virus liquid obtained by suspension culture. Since a large amount of foreign proteins exist in the virus liquid obtained by suspension culture, multi-step purification is usually required to effectively remove the foreign proteins, but multi-step purification will increase virus loss and reduce virus immunogenicity. During the research and development process, the present invention unexpectedly found that the bovine parainfluenza virus type 3 virus liquid obtained by suspension culture was purified and concentrated using a hollow fiber column, and diafiltration was performed using a specific gradient diafiltration volume, which can significantly improve the removal efficiency of foreign proteins while ensuring a high virus recovery rate.

[0006] Specifically, the present invention provides the following technical solutions: The present invention provides a method for purifying and concentrating bovine parainfluenza virus type 3, the method comprising: purifying and concentrating a virus solution of bovine parainfluenza virus type 3 by using a hollow fiber column; The purification and concentration includes three diafiltrations, and the volumes of diafiltration liquid used in the three diafiltrations decrease in sequence, wherein the volume of diafiltration liquid used in the first diafiltration is 5-10 times the concentrated volume, the volume of diafiltration liquid used in the second diafiltration is 2-5 times the concentrated volume, and the volume of diafiltration liquid used in the third diafiltration is 1-2 times the concentrated volume.

[0007] In the above method, the volume of diafiltration liquid used for the three diafiltrations decreases successively, which means that the volume of diafiltration liquid used for the first diafiltration is greater than the volume of diafiltration liquid used for the second diafiltration, and the volume of diafiltration liquid used for the second diafiltration is greater than the volume of diafiltration liquid used for the third diafiltration.

[0008] The present invention finds that the use of a hollow fiber column in combination with the above-mentioned diafiltration volume gradient for diafiltration can significantly improve the removal efficiency of foreign proteins while ensuring a high virus recovery rate.

[0009] As a preferred embodiment of the present invention, the volume of the diafiltration liquid used in the first diafiltration is 5-6 times the concentrated volume, the volume of the diafiltration liquid used in the second diafiltration is 2-3 times the concentrated volume, and the volume of the diafiltration liquid used in the third diafiltration is 1-1.5 times the concentrated volume.

[0010] As another preferred embodiment of the present invention, the volume of the diafiltration liquid used in the first diafiltration is 9-10 times the concentrated volume, the volume of the diafiltration liquid used in the second diafiltration is 5-6 times the concentrated volume, and the volume of the diafiltration liquid used in the third diafiltration is 2-3 times the concentrated volume.

[0011] In the above method, the above three diafiltrations are performed after concentration to the final volume, and after the three diafiltrations, the concentrated liquid is collected to obtain a purified and concentrated bovine parainfluenza virus type 3 virus liquid.

[0012] In the above method, the concentration volume is the volume of the concentrated virus solution obtained at the end of purification and concentration.

[0013] In the above method, the washing filtrate is PBS.

[0014] Preferably, the concentration of the PBS is 0.1-0.15 mol / L. Using PBS in this concentration range for diafiltration can more effectively remove foreign proteins.

[0015] Preferably, the pH of the PBS is 7.3-7.4.

[0016] Preferably, the molecular weight cut-off of the hollow fiber column is 500-750KD.

[0017] In the above method, the concentration multiple of the purification and concentration is preferably 5-10 times.

[0018] A person skilled in the art can adjust and select a suitable concentration multiple within the above range according to the volume of the initial virus liquid, and calculate the volume of the final concentrated virus liquid according to the volume of the initial virus liquid and the concentration multiple.

[0019] In the present invention, the virus liquid is the virus liquid obtained by suspension culture. The virus liquid obtained by suspension culture is the virus culture liquid collected at the end of suspension culture.

[0020] Preferably, the protein content of the virus liquid obtained by suspension culture is 1-10 mg / mL; preferably 3-9 mg / mL.

[0021] More preferably, the TCID of the virus solution obtained by suspension culture is 50 / 0.1mL is 6-8, and the protein content is 6-9mg / mL.

[0022] The above method also includes: before purification and concentration, clarifying the virus liquid obtained by suspension culture.

[0023] The purpose of clarification is to remove cell debris and some impurities. However, the removal effect of clarification on impurities is limited, and the clarified virus fluid still contains a lot of impurities.

[0024] Preferably, the clarification treatment is to centrifuge the virus solution obtained by suspension culture.

[0025] Preferably, the clarified virus solution should be kept at 4-6°C.

[0026] In some embodiments of the present invention, the purification and concentration method comprises the following steps: (1) Centrifuging the bovine parainfluenza virus type 3 fluid obtained from suspension culture to remove cell debris and some impurity proteins to obtain a clarified viral fluid; (2) The clarified virus solution is purified and concentrated 5 to 10 times using a 500-750KD hollow fiber column. After concentration to the final volume, three diafiltrations are performed using different diafiltration volumes, wherein the volume of the diafiltration solution used in the first diafiltration is 5 to 10 times the concentrated volume, the volume of the diafiltration solution used in the second diafiltration is 2 to 5 times the concentrated volume, and the volume of the diafiltration solution used in the third diafiltration is 1 to 2 times the concentrated volume; the diafiltration solution is PBS with a concentration of 0.1 to 0.15 mol / L and a pH of 7.3 to 7.4.

[0027] The virus liquid concentrated by the above purification has different levels of improvement in virus titer according to the concentration multiples (for example, the virus titer of the virus liquid concentrated 5 times is increased by 0.5 titer, and the virus titer of the virus liquid concentrated 10 times is increased by 1 titer), and the removal rate of impurity proteins can reach more than 90%.

[0028] The present invention also provides bovine parainfluenza virus type 3 concentrated virus liquid prepared by the method.

[0029] The beneficial effects of the present invention include at least: the purification and concentration method of bovine parainfluenza virus type 3 provided by the present invention can effectively remove foreign proteins (the foreign protein removal rate reaches more than 90%) for the bovine parainfluenza virus type 3 virus liquid in suspension culture, and has a high virus recovery rate, can significantly improve the toxicity of the virus liquid, and the foreign protein content of the concentrated virus liquid is greatly reduced. At the same time, compared with three large-volume filtrations, the filtration time can be effectively saved. The vaccine prepared as an immunogen has no local and systemic adverse reactions after immunizing animals, and has good immunogenicity. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example 1

[0032] This embodiment provides a method for purifying and concentrating bovine parainfluenza virus type 3, comprising the following specific steps: 1. Suspend the cultured bovine parainfluenza virus type 3 virus solution (TCID 50 / 0.1mL is 6.75, the protein content is 7.63mg / mL) 5000mL, and the clarified virus liquid is obtained by centrifugation in a continuous flow centrifuge, wherein the centrifugal speed is 8000r / min, the centrifugal time is 30min, and the TCID of the clarified virus liquid is 50 / 0.1mL is 7.0, the protein content is 7.55mg / mL, and the protein removal rate is 1.05%.

[0033] 2. The clarified virus solution was purified and concentrated 10 times to 500 mL by a 500KD hollow fiber column; after concentration to the final volume, the transmembrane pressure was maintained at 7-8 psi and the circulation flow rate was 300 mL / min. 2500 mL, 1000 mL, and 500 mL of PBS (concentration of 0.1 mol / L, pH 7.3) were used for three times of diafiltration to obtain a purified and concentrated virus solution with a final concentration of 10 times. The TCID 50 / 0.1mL was 7.75, the protein content was 0.26mg / mL, the protein removal rate was 96.72%, and the virus recovery rate was 100%.

[0034] Example 2

[0035] This embodiment provides a method for purifying and concentrating bovine parainfluenza virus type 3, comprising the following specific steps: 1. Suspend the cultured bovine parainfluenza virus type 3 virus solution (TCID 50 / 0.1mL is 7.25, protein content is 6.71mg / mL) 500L, and the clarified virus liquid is obtained by continuous flow centrifugation. The TCID 50 / 0.1mL was 7.25, the protein content was 6.34mg / mL, and the protein removal rate was 5.51%.

[0036] 2. The clarified virus solution was purified and concentrated 10 times to 50L through a 750KD hollow fiber column, the transmembrane pressure was maintained at 0.02~0.04bar, the circulation flow rate was 80L~100L / min, and the filtration was performed three times with PBS (concentration of 0.1mol / L, pH 7.4) 500L, 250L, and 100L, respectively, to obtain a purified and concentrated virus solution with a final concentration of 5 times. The TCID of the concentrated virus solution 50 / 0.1mL was 7.75, the protein content was 0.57mg / mL, the protein removal rate was 91.50%, and the virus recovery rate was 99.9%.

[0037] Comparative Example 1

[0038] This comparative example provides a method for purifying and concentrating bovine parainfluenza virus type 3, comprising the following specific steps: 1. Suspend the cultured bovine parainfluenza virus type 3 virus solution (TCID 50 / 0.1mL is 6.75, protein content is 7.63mg / mL) 5000mL, and the clarified virus liquid is obtained by centrifugation in a continuous flow centrifuge. The centrifugal speed is 8000r / min, the centrifugal time is 30min, and the TCID of the clarified virus liquid is 50 / 0.1mL is 7.0, the protein content is 7.55mg / mL, and the protein removal rate is 1.05%.

[0039] 2. The clarified virus solution was purified and concentrated 10 times to 500 mL (final volume) through a 500KD hollow fiber column. After concentration to the final volume, the transmembrane pressure was maintained at 7-8 psi and the circulation flow rate was 300 mL / min. 500 mL, 500 mL, and 500 mL of PBS (concentration of 0.1 mol / L, pH 7.3) were used for three times of diafiltration to obtain a purified and concentrated virus solution with a final concentration of 10 times. The TCID 50 / 0.1mL was 7.75, the protein content was 5.56mg / mL, the protein removal rate was 27.52%, and the virus recovery rate was 100%.

[0040] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for purifying and concentrating bovine parainfluenza virus type 3, characterized in that: The method comprises: purifying and concentrating a virus solution of bovine parainfluenza virus type 3 using a hollow fiber column; The purification and concentration includes three diafiltrations, and the volumes of diafiltration liquid used in the three diafiltrations decrease in sequence, wherein the volume of diafiltration liquid used in the first diafiltration is 5-10 times the concentrated volume, the volume of diafiltration liquid used in the second diafiltration is 2-5 times the concentrated volume, and the volume of diafiltration liquid used in the third diafiltration is 1-2 times the concentrated volume.

2. The method for purifying and concentrating bovine parainfluenza virus type 3 according to claim 1, characterized in that: The volume of the diafiltration liquid used in the first diafiltration is 5-6 times the concentrated volume, the volume of the diafiltration liquid used in the second diafiltration is 2-3 times the concentrated volume, and the volume of the diafiltration liquid used in the third diafiltration is 1-1.5 times the concentrated volume; Alternatively, the volume of the diafiltration liquid used in the first diafiltration is 9-10 times the concentrated volume, the volume of the diafiltration liquid used in the second diafiltration is 5-6 times the concentrated volume, and the volume of the diafiltration liquid used in the third diafiltration is 2-3 times the concentrated volume.

3. The method for purifying and concentrating bovine parainfluenza virus type 3 according to claim 1 or 2, characterized in that: The washing filtrate is PBS; Preferably, the concentration of the PBS is 0.1-0.15 mol / L.

4. The method for purifying and concentrating bovine parainfluenza virus type 3 according to claim 3, characterized in that: The pH of the PBS is 7.3-7.

4.

5. The method for purifying and concentrating bovine parainfluenza virus type 3 according to any one of claims 1 to 4, characterized in that: The molecular weight cut-off of the hollow fiber column is 500-750KD.

6. The method for purifying and concentrating bovine parainfluenza virus type 3 according to any one of claims 1 to 5, characterized in that: The concentration multiple of the purification and concentration is 5-10 times.

7. The method for purifying and concentrating bovine parainfluenza virus type 3 according to any one of claims 1 to 6, characterized in that: The virus liquid is obtained by suspension culture.

8. The method for purifying and concentrating bovine parainfluenza virus type 3 according to claim 7, characterized in that: The protein content of the virus liquid obtained by suspension culture is 1-10 mg / mL; preferably 3-9 mg / mL.

9. The method for purifying and concentrating bovine parainfluenza virus type 3 according to claim 7 or 8, characterized in that: The method further comprises: before purification and concentration, clarifying the virus liquid obtained by suspension culture.

10. The method for purifying and concentrating bovine parainfluenza virus type 3 according to claim 9, characterized in that: The clarification treatment is to centrifuge the virus solution obtained through suspension culture.